KIT QUANTA - standardization kit for absolute protein quantitation: study of the digestion kinetic of the chimeric protein standard

In the world of biomarker discovery, up to now, only a few biomarkers have been validated. The validation remains a crucial step in the development of a biomarker but is quite challenging and lengthy. In this context, the development of high-throughput techniques is therefore needed. Mass spectrometry-based methods such as SRM has become a prominent choice for large-scale protein quantification. The difficulty of this approach lies in the implementation of standardization for absolute quantification. Different standardization approaches now exist in proteomics but all of them have their limitation. One example is the use of isotopically-labelled peptides for the absolute quantification of proteins. This method is highly specific of the protein to quantify but does not take into account all the bias introduced at each step of the process applied before SRM analyses. In this context, we are developing a comprehensive standardization strategy aiming to control the entire sample preparation process and easing the performance of longitudinal or large-scale studies of biomarker validation. Combined to the isotopically-labelled peptides strategy, we built a chimeric protein containing a structured part associated to an intrinsically disordered domain that will be used, among other things, as a control of the performance of the digestion protocol. The principle is to monitor the accessibility of cleavage sites in function of the structural state of the protein and the digestion protocol applied. For this purpose, digestion kinetics were performed on this protein to analyse the gradual release of the peptides depending on their conformational environment within the protein.